共查询到20条相似文献,搜索用时 15 毫秒
1.
A simple procedure for maximum yield of high-quality plasmid DNA 总被引:40,自引:0,他引:40
We have established a simple procedure for the rapid isolation of high-quality plasmid DNA suitable for various molecular techniques and provided a step-by-step protocol. The DNA samples isolated by this procedure have been used successfully for double-stranded DNA sequencing, restriction enzyme mapping, subcloning, in vitro mutagenesis, generation of deletion clones and so on. The procedure is highly reproducible, and superior quality DNA can be obtained without the use of phenol, chloroform or other organic solvents. 相似文献
2.
D K Lahiri S Bye J I Nurnberger M E Hodes M Crisp 《Journal of biochemical and biophysical methods》1992,25(4):193-205
We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells. 相似文献
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Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol 总被引:6,自引:0,他引:6
We have developed a new, rapid method for the extraction of human genomic DNA from whole blood samples. Traditionally, genomic DNA has been extracted from blood by overnight proteinase K digestion of lysed peripheral lymphocytes followed by phenol/chloroform extraction. In addition to being time consuming, the use of phenol involves inherent risks due to the toxic nature of the reagent. Our method for the extraction of DNA from whole blood uses sodium perchlorate and chloroform instead of phenol with a significant time savings realized as well as fewer hazards to the technician. Furthermore, DNA prepared by this new method is an excellent substrate for restriction endonuclease digestion and Southern hybridization analysis. 相似文献
5.
Dichloromethane as an economic alternative to chloroform in the extraction of DNA from plant tissues
Alba Lucía Chaves Claudia E. Vergara Jorge E. Mayer 《Plant Molecular Biology Reporter》1995,13(1):18-25
Processing of large numbers smaples of plant tissue samples for molecular mapping and gene tagging requires methods that are
quick, simple, and cheap, and that eventually can be automated. Organic solvents used for DNA extraction can represent a significant
proportion of the overall cost. In this study we examined dichloromethane as a replacement for chloroform to be used in combination
with phenol. 相似文献
6.
Ariel Louwrier 《Biotechnology Techniques》1999,13(5):329-330
Nucleic acids were extracted from bacteria and yeast, using a biphasic system created by mixing a water-miscible, organic solvent with an aqueous salt solution. Nucleic acids were separated from the majority of host-contaminating proteins without using chaotrophic agents such as guanidine salts, phenol or chloroform, which are known to be hazardous. Isolated DNA is sufficiently pure for use in the polymerase chain reaction (PCR). 相似文献
7.
Isolation of polysaccharide-free DNA from plants 总被引:2,自引:2,他引:0
Benoît Rether Geneviève Delmas Abdelkrim Laouedj 《Plant Molecular Biology Reporter》1993,11(4):333-337
A quick procedure for the isolation of polysaccharide-free DNA from different plant species and cell suspension or callus
cultures is described. The originality of the method lies in the use of a mixture of glycoside hydrolases that leads, after
phenol and chloroform extraction, to the isolation of pure DNA without any polysaccharide contamination. The highly purified
DNA can be used for nucleotide analysis by HPLC, RFLP analysis and PCR amplification. 相似文献
8.
Whitney L. E. Tsai Margaret E. Schedl James M. Maley John E. McCormack 《Molecular ecology resources》2020,20(5):1220-1227
Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols. 相似文献
9.
Simple and Safe Method for Simultaneous Isolation of Microbial RNA and DNA from Problematic Populations
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Simon McIlroy Kate Porter Robert J. Seviour Daniel Tillett 《Applied microbiology》2008,74(21):6806-6807
We describe a novel, rapid, and safe method for extracting RNA and DNA from refractory microbes, which avoids the use of phenol or chloroform. It has been used successfully to isolate high-quality nucleic acids from pure cultures and environmental populations known to resist widely used extraction protocols. 相似文献
10.
Extremely Rapid Extraction of DNA from Bacteria and Yeasts 总被引:22,自引:0,他引:22
A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is
presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform
to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction
enzyme digestion and genomic library construction. This method is reproducible and simple for the routine DNA extraction from
bacteria and yeasts. 相似文献
11.
Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory.
This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified
DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature
agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic
solvents such as phenol. 相似文献
12.
We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid. 相似文献
13.
To remove or inactivate an enzyme from DNA in multistep procedures in molecular biology, it is often necessary to phenol extract the solution, followed by chloroform extraction and ethanol precipitation. In addition to being time-consuming and hazardous, there can be significant loss of DNA with this procedure, especially when small volumes or amounts of DNA are being used. We have found that filtering analytical reaction mixtures through a hydrophobic membrane specifically to remove protein is a rapid alternative to phenol extraction. Within broad limits commonly encountered in molecular biology, filtration through a polyvinylidene difluoride membrane quantitatively removes a variety of enzymes without significant loss of double-stranded nucleic acid. 相似文献
14.
A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay
Lockhart MG Graves SR Banazis MJ Fenwick SG Stenos J 《Letters in applied microbiology》2011,52(5):514-520
Aims: To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small‐cell variant (SCV) by real‐time PCR (qPCR) in clinical samples. Methods and Results: A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method. Conclusions: The silica column extraction method was more efficient at recovering C. burnetii DNA, from large‐cell and small‐cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples. Significance and impact of study: This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV. 相似文献
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Tobias Kieser 《Plasmid》1984,12(1):19-36
Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids. 相似文献
17.
Dixson JD 《Hereditas》2005,142(2005):22-23
DNA isolation is often the limiting step in genetic analysis using PCR and automated fragment analysis due to low quality or purity of DNA, the need to determine and adjust DNA concentrations after isolation etc. Several protocols have been developed which are either safe and provide good quality DNA or hazardous and provide excellent quality DNA. In this brief communication I describe a new and rapid method of DNA isolation which employs the co-precipitation of protein and polyester, in the presence of acetone, to remove contaminating proteins from a lysed-tissue sample, thus leaving high quality pure DNA. The advantages of this method are increased safety over the phenol:chloroform and the chaotrophic salt methods and increased purity over the salting-out method. Since the concentrations of DNA isolated using this method are relatively consistent regardless of the amount of starting tissue (within limits), adjustments of the DNA concentrations before use as templates in PCR's are not necessary. 相似文献
18.
A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks. 相似文献
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Byung‐Hyuk Kim Hee‐Mock Oh Young‐Ki Lee Gang‐Guk Choi Chi‐Yong Ahn Byung‐Dae Yoon Hee‐Sik Kim 《Journal of phycology》2006,42(5):1137-1141
A simple and rapid method is presented for the preparation of RNA from various cyanobacteria. Unlike other methods that require a lysis solution, lysozymes, or proteinase K, the proposed method, called the bead–phenol–chloroform (BPC) method, uses silica/zirconia beads, phenol, and chloroform to break the cells and extract RNA more efficiently. Experiments confirm that the BPC method can successfully isolate total RNA from various cyanobacterial strains without DNA contamination, and the extracted RNA samples have a relatively high purity, concentration, and yield. Furthermore, the BPC method is more rapid, simple, and economical when compared with previously reported methods. 相似文献